Microfluidics and lab-on-a-chip hold the promise of miniaturized biological and chemical testing [1], and many microfluidic systems have been demonstrated for the purposes of electrophoresis [1], genetic analysis [2], and chemiluminescence assays [3]. One additional technique that is commonly used in such systems is fluorescence. When illuminated with light of a particular color (the excitation wavelength), fluorescent materials luminesce in a different color (the emission color). The intensity of the emitted light relates to the concentration of the fluorescent dye. This is the most commonly used approach in the mainstream biological analyses.
Fluorescence detection systems, however, have proven difficult to miniaturize [4], though many different systems have been proposed [5,6]. See also, U.S. Pat. Nos. 7,800,753; 7,525,653; 7,795,014; 7,709,249; 7,659,968; 7,653,429; 7,569,382; 7,563,350; 7,550,069; 7,531,363; 7,515,953; 7,511,811; 6,995,841; 7,507,575; 7,463,353; 7,444,053; 7,142,303 6,686,201; 6,038,023; and 5,784,157, and US Published Patent Application Nos. 2002/0109844, 2010/0032582, each of which is expressly incorporated herein by reference. Fluorescence measurements typically require optical sources, lenses and detectors, and these components are difficult to miniaturize and integrate effectively on a lab-on-a-chip. Typically, optical filters are used to shield the detector or sensor, and these optical filters have to be specifically matched to the excitation light and the dye. For example, the Rhodamine 6G-specific filter used in a fluorescence microscope shields excitation (green) light but passes emission (red) light through to the eyepiece.
Shown in FIG. 1 is a polarization-based optical isolation technique which has recently been demonstrated [7,8]. It has the advantage of being color-independent, and as such does not require different filters for different colors. Here, the excitation light is polarized before being incident on the dye, and a cross-polarized filter after the dye blocks most of the excitation light from the filter. The emission light, which is un-polarized, is then detected.
In its simplest implementation, it can only quantify a single dye at a time, since the sensor cannot discriminate the color of the incident light.
The application of fluorescence detection techniques in conjunction with microelectromechanical systems (MEMS) including types of lab-on-a-chip is known. See, e.g., U.S. Pat. Nos. 7,800,753; 7,797,988; 7,788,972; 7,741,618; 7,723,116; 7,722,753; 7,715,001; 7,708,873; 7,692,783; 7,691,244; 7,645,596; 7,630,073; 7,612,883; 7,598,371; 7,590,161; 7,586,604; 7,531,363; 7,525,653; 7,476,504; 7,465,382; 7,463,353; 7,461,547; 7,444,053; 7,302,830; 7,291,564; 7,259,217; 7,215,425; 7,163,658; 7,087,444; 7,016,022; 6,986,739; 6,934,836; 6,934,435; 6,887,202; 6,881,979; 6,846,638; 6,762,025; 6,716,948; 6,685,810; 6,682,942; 6,608,360; 6,596,545; 6,379,929; 6,331,439; 6,287,765; 6,118,126; 5,980,704; 5,603,351, and US Published Patent Application Nos. 2010/0264032; 20100240044; 2010/0230613; 2010/0210008; 2010/0152066; 2010/0105035; 2010/0101411; 2010/0097594; 2010/0075438; 2009/0279093; 2009/0257920; 2009/0170118; 2009/0131858; 2009/0107844; 2009/0098541; 2009/0090174; 2009/0078036; 2009/0042744; 2009/0002700; 2008/0285039; 2008/0255458; 2008/0165346; 2008/0157005; 2008/0145278; 2008/0047836; 2008/0000772; 2007/0279631; 2007/0279626; 2007/0240989; 2007/0206187; 2007/0188750; 2007/0183928; 2007/0163663; 2007/0154881; 2007/0134128; 2007/0128615; 2007/0122314; 2007/0090026; 2007/0031861; 2006/0251371; 2006/0243047; 2006/0231771; 2006/0191793; 2006/0183145; 2006/0128917; 2006/0078472; 2006/0033910; 2005/0182307; 2005/0164264; 2005/0147976; 2005/0089890; 2005/0088653; 2005/0084203; 2005/0079526; 2005/0014134; 2004/0253365; 2004/0214177; 2004/0152076; 2004/0080011; 2004/0076946; 2004/0072356; 2004/0005769; 2004/0005582; 2003/0225362; 2003/0138973; 2003/0100824; 2003/0052007; 2003/0052006; 2003/0000291; 2002/0176804; 2002/0172969; 2002/0168671; 2002/0164824; 2002/0110932; 2002/0074553; 2002/0058273; 2002/0034827; 2002/0034757; 2002/0026937, each of which is expressly incorporated herein by reference.
See also, K. G. Libbrecht, E. D. Black, and C. M. Hirata, “A basic lock-in amplifier experiment for the undergraduate laboratory” Am. J. Phys. 71, pp. 1208. authors.library.caltech.edu/12641/1/LIBajp03.pdf; Joseph R. Lakowicz, Principles of fluorescence spectroscopy, Springer, 2006, p. 743; P. Herman, et al., “Frequency Domain Fluorescence Microscopy with the LED as a Light Source”, J. Microscopy 203, part 2, August 2001, pp. 176-181; A. Zukauskas, N. Kurilcik, P. Vitta, S. Jursenas, E. Bakien, R. Gaska, “Optimization of a UV light-emitting diode based fluorescence-phase sensor”, Proc. SPIE, Vol. 6398, 63980Y (2006); doi:10.1117/12.689907; T. L. Chester and J. D. Winefordner, “Evaluation of the analytical capabilities of frequency modulated sources in multielement non-dispersive flame atomic fluorescence spectrometry”, Spectrochimica Acta Part B: Atomic Spectroscopy, Volume 31, Issue 1, 1976, Pages 21-29, doi:10.1016/0584-8547(76)80003-1; U. Schreiber, “Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer”, Photosynthesis Research, Volume 9, Numbers 1-2, 261-272, DOI: 10.1007/BF00029749.